Field of the Invention
An antimicrobial formulation containing a mixture of organic acids, aldehydes and organic acid esters, where such combination results in a synergistic response.
Background
The Centers for Disease Control and Prevention (CDC) estimates that roughly one out of six Americans or 48 million people is sickened by food borne illnesses each year. Another 128,000 are hospitalized and approximately 3,000 die of food borne disease every year. In 2011, the CDC (http://www.cdc.gov/outbreaknet/foodborne-surveillance-questions-and-answers.html) estimated that salmonellosis resulted in 20,000 hospitalizations and 378 cases of death per year. It has also estimated that Escherichia coli O157:O7 causes approximately 62,000 cases of food borne disease and approximately 1,800 food borne illness-related hospitalizations in the United States annually. A study by the Pew Charitable Trusts of Georgetown University suggested that food borne illnesses cost the United States $152 billion in health-related expenses each year (Yeager, 2010).
A study commissioned by the UK Food Standard Agency (FSA) found that campylobacter was one of the main causes of Infectious Intestinal Diseases (IID) and was responsible for around 500,000 cases annually. The same agency also reported that two thirds of chicken samples on sale within the UK were contaminated with campylobacter (http://www.food.gov.uk/policy-advice/microbiologykampylobacterevidenceprogramme/campybackground).
The world's tendency to find more natural and/or organic antimicrobials has resulted in a great amount of research in identifying these type of products as well as an increased cost of new raw materials due to low commercial availability of natural/organic products. Currently many type of chemicals and their combinations are used as antimicrobials. These chemicals include organic acids, aldehydes, ester of organic acids, plant extracts and others.
One of the components of the present invention are organic acid esters. Several US patents and WO patents described the use of organic acid esters as flavorings, preservatives or antimicrobials. U.S. Pat. No. 7,652,067 and WO Patent #2009/037270 suggest of the use of a hydrophobic organic compound i.e. menthol, with a monoester of a saturated organic acid of C6-C20 carbon length. This product is useful for flavoring food and perfumery. These patents do not suggest of a combination of organic acid esters combined with organic acids and aldehydes as antimicrobials. US Patent Application #2009/0082253, suggests of an antimicrobial comprising a mixture of organic acid esters of lactic acid (lactylate), a hydroxyl carboxylic acid and an antibacterial agent. They do not suggest that the mixture of esters of organic acids other than lactic acids ester and polylysine, a known antimicrobial, will result in an effective antimicrobial. U.S. Pat. No. 7,862,842 suggest the use of organic acid ethyl esters derived from lauric acid and arginine preservative for perishable food product not as animal feed preservative.
The present invention suggests the use of organic acid esters in combination with aldehydes and organic acids as an antimicrobial in feed ingredients, feed and water. Literature review has shown that organic acid esters have been studied as bactericides and fungicides against plant and human pathogens. Propyl, methyl and ethyl esters of ferulic acid were effective in inhibiting Saccharomyces cerevisiae, Aspergillus fumigatus and Aspergillus flavus (Beck, et. al, 2007). Organic acids esters prepared from mixing n-organic alcohols and dibasic acids were used as plasticizer and exhibited some benefits as a fungicide (Sadek, et. al., 1994). Six organic acid esters from soybean, including methyl and ethyl palmitates, methyl and ethyl oleates, methyl linoleate and methyl linolenate demonstrated curative and protective activities against powdery mildew in barley. Methyl laurate has also been reported to control the development of powdery mildew (Choi, et. al., 2010). Castor oil methyl ester can replace mineral oil to control the fungal disease, Black Sigatoka, in bananas (Madriz-Guzman, et. al., 2008). Organic acid methyl esters from linoleic, linolenic, arachidonic, palmitoleic and oleic acids were effective in inhibiting growth of Streptococcus mutans, Candida albicans, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum and Porphyromonas gingivalis (Huang, et. al., 2010). The fungus Muscodor albus produces certain volatiles compounds that effectively inhibit and kill other fungi and bacteria. One of these volatile compounds is an ester of 1-butanol, 3-methyl acetate, which is 62% of the total esters that was effective in inhibit growth of several fungi (Strobel, et. al., 2001). The organic acid methyl ester profile from Sesuvium portulacastrum indicates the presence of palmitic, oleic, linoleic, linolenic, myristic and beheni acid esters, all of them effective against several human pathogenic microorganisms (Chandrasekaran, et. al., 2011). Organic acid methyl esters of dodecanoic and pentadecanoic acids found in carrots extract were effective against Leuconostoc mesenteroides, Listeria monocytogenes, Staphylococcus aureus, Pseudomonas fluorescens, Candida albicans and E. coli (Babic, et. al., 1994). The inhibitory activity against E. coli, L. monocytogenes, Fusarium culmorum, Bacillus cereus and Saccharomyces cerevisiae was higher when using phenolic acid butyl esters than methyl esters (Merkl, et. al., 2010).
Another compound of the present invention is an aldehyde. One of the most effective of these aldehydes, formaldehyde, has been used as an antiseptic for many years. Two U.S. Pat. Nos. 5,547,987 and 5,591,467 suggest the use of formaldehyde to control Salmonella spp. in animal feed. These patents do not suggest that the combination of formaldehyde or other aldehydes with organic acid esters provides a synergistic effect as described in the present invention.
An aldehyde used in the present invention is trans-2-hexenal, a six carbon, double bond aldehyde, C6H10O and MW=98.14. Trans-2-hexenal is present in many edible plants such as apples, pears, grapes, strawberries, kiwi, tomatoes, olives, etc. The use of plants and plant extracts have been successful in identifying new anti-microbials. For example, the extract from cashew apple was observed to effective against Helicobacter pylori and S. cholerasuis at concentrations of 50-100 ug/ml. The two main components were found to be anacardic acid and trans-2-hexenal. The minimum inhibitory and minimum biocidal activity of trans-2-hexenal were determined to be 400 and 800 ug/ml, respectively (Kubo, et. al., 1999; Kubo and Fujita, 2001). Kim and Shin (2004) found that trans-2-hexenal (247 mg/L) was effective against B. cereus, S. typhimurium, V. parahaemolyticus, L. monocytogenes, S. aureus and E. coli O157:H7. Nakamura and Hatanaka (2002) demonstrated that trans-3-hexenal was effective in controlling Staphylococcus aureus, E. coli and Salmonella typhimurium at a level of 3-30 ug/ml. Trans-2-hexenal completely inhibited proliferation of both P. syringae pathovars (570 μg/L of air) and E. coli (930 micrograms/L of air)(Deng, et. al., 1993). It was observed that trans-2-hexenal at 250 ug/ml was effective on inhibiting the growth of Phoma mycelium (Saniewska and Saniewski, 2007). In a study to control mold in fruits, it was found that trans-2-hexenal was not phytotoxic to apricots, but it was phytotoxic for peaches and nectarines at 40 μl/l (Neri, et. al., 2007). Trans-2-hexenal (12.5 μl/l) was effective on controlling Penicillium expansum that causes blue mold (Neri, et. al., 2006a and 2006b). Fallik et. al. (1998) and Hamilton-Kemp et. al. (1991), suggested that trans-2-hexenal vapors inhibited the germination of Botrytis spores and apple pollen.
USPTO Application #2007/0087094 suggests the use of at least two microbiocidally active GRAS compounds in combination with less than 50% alcohol (isopropanol or isopropanol/ethanol) as a microbicide. Trans-2-hexenal could be considered one of the GRAS compounds (USPTO Application No. 2007/0087094). Archbold, et. al. (1994) observed that the use of trans-2-hexenal at 0.86 or 1.71 mmol (100 or 200 microliters neat compound per 1.1 L container, respectively) for 2 weeks as for postharvest fumigation of seedless table showed promise for control of mold.
U.S. Pat. No. 5,698,599 suggests a method to inhibit mycotoxin production in a foodstuff by treating with trans-2-hexenal. Trans-2-hexenal completely inhibited the growth of A. flavus, P. notatum, A. alternate, F. oxysporum, Cladosporium spp., B. subtilis and A. tumerfaciens at a concentration of 8 ng/l air. When comparing trans-2-hexenal to citral for the control of yeast (105 CFU/bottle) in beverages it was found that 25 ppm of trans-2-hexenal and thermal treatment (56° C. for 20 min) was equivalent to 100-120 ppm citral. In beverages that were not thermally treated, 35 ppm of trans-2-hexenal was necessary to control microorganisms (Belleti, et. al., 2007). Trans-2-hexenal has also been reported to control insects, such as Tibolium castaneum, Rhyzopertha dominica, Sitophilus granaries, Sitophilus orazyzae and Cryptolestes perrugineus (Hubert, et. al., 2008). U.S. Pat. No. 6,201,026 suggests of an organic aldehyde of 3 or more carbons for the control of aphides.
Several patents suggest the use of trans-2-hexenal as a fragrance or perfume. U.S. Pat. No. 6,596,681 suggests the use of trans-2-hexenal as a fragrance in a wipe for surface cleaning. U.S. Pat. No. 6,387,866, U.S. Pat. No. 6,960,350 and U.S. Pat. No. 7,638,114 suggest the use of essential oil or terpenes (e.g. trans-2-hexenal) as perfume for antimicrobial products. U.S. Pat. No. 6,479,044 demonstrates an antibacterial solution comprising an anionic surfactant, a polycationic antibacterial and water, where an essential oil is added as perfume. This perfume could be a terpene such as trans-2-hexenal or other type of terpenes. U.S. Pat. No. 6,323,171, U.S. Pat. No. 6,121,224 and U.S. Pat. No. 5,911,915 demonstrate an antimicrobial purpose microemulsion containing a cationic surfactant where an essential oil is added as a perfume. This perfume can be various terpenes including i.e. trans-2-hexenal. U.S. Pat. No. 6,960,350 demonstrates an antifungal fragrance where a synergistic effect was found when different terpenes were used in combinations (for example trans-2-hexenal with benzaldehyde).
The mode of action of trans-2-hexenal is thought to be the alteration of the cell membrane due to the reaction of hexenal to the sulfhydryl moiety or cysteine residues or formation of Schiff bases with amino groups of peptides and proteins (Deng, et. al., 1993). Trans-2-hexenal is reported to act as a surfactant, but likely permeates by passive diffusion across the plasma membrane. Once inside cells, its α,β-unsaturated aldehyde moiety reacts with biologically important nucleophilic groups. This aldehyde moiety is known to react with sulphydryl groups mainly by 1,4-additions under physiological conditions (Patrignani, et. al., 2008).
Trans-2-hexenal is an inhibitor of phospholipase D, an enzyme that catalyses the hydrolysis of membrane phospholipids that occurs during the maturation and ripening of many types of fruits and vegetables. Therefore, it is suggested that trans-2-hexenal may inhibit ripening (USPTO Application No. 2005/0031744 A1). It is suggested that the inhibition of Salmonella typhimurium and Staphylococcus aureus by trans-2 hexenal is due to the hydrophobic and hydrogen bonding of its partition in the lipid bilayer. The destruction of electron transport systems and the perturbation of membrane permeability have been suggested as other modes of action (Gardine, et. al., 2001). The inhibition of P. expansum decay may be due to damage to fungal membranes of germinating conidia (Neri, et. al., 2006a and 2006b). Studies have been performed to compare trans-2-hexenal to other similar compounds. Deng, et. al. (1993) showed that unsaturated volatiles, trans-2-hexenal and trans-2-hexen-1-ol, exhibited a greater inhibitory effect than the saturated volatiles, hexanal and 1-hexanol. Trans-2-hexenal was more active than hexanal, nonanal and trans-2-octenal against all ATCC bacterial strains (Bisignano, et. al., 2001). Other have found that trans-2-hexenal had lower minimal fungal-growth-inhibiting concentrations than hexanal, 1-hexanol, trans-2-hexen-1-ol, and (Z)-3-hexen-1-ol (basically aldehydes>ketones>alcohols; Andersen, et. al., 1994). Hexenal and hexanoic acid have been reported to be more effective than hexanol in inhibiting Salmonella spp. (Patrignani, et. al., 2008).
Muroi, et. al., (1993) suggested that trans-2-hexenal exhibited broad antimicrobial activity but its biological activity (50 to 400 μg/ml) is usually not potent enough to be considered for practical applications. Studies have shown that trans-2-hexenal can potentiate the effectiveness of certain type of antimicrobials. Several patents suggest the use of potentiators for aminoglycoside antibiotics (U.S. Pat. No. 5,663,152), and potentiators for polymyxin antibiotic (U.S. Pat. No. 5,776,919 and U.S. Pat. No. 5,587,358). These potentiators can include indol, anethole, 3-methylindole, 2-hydroxy-6-R-benzoic acid or 2-hexenal. A strong synergic effect was observed when trans-2-eptenal, trans-2-nonenal, trans-2-decenal and (E,E)-2,4-decadienal were tested together (1:1:1:1 ratio) against ATCC and clinically isolated microbial strains (Bisignano et. al., 2001). The prior art has not suggested or observed that the use of trans-2-hexenal in combination with organic acids esters improved the antimicrobial activity of either of the components by themselves
Another component of the present invention are organic acids. Commercial mold inhibitors and bactericides are composed of single organic or a mixture of organic acids and/or formaldehyde. The most commonly used acids are propionic, benzoic acid, butyric acid, acetic, and formic acid. The mechanism by which small chain organic acids exert their antimicrobial activity is that undissociated (RCOOH=non ionized) acids are lipid permeable and in this way they can cross the microbial cell wall and dissociate in the more alkaline interior of the microorganism (RCOOH→RCOO−+H+) making the cytoplasm unstable for survival (Van Immerseel, et. al., 2006; Paster, 1979).
Nonanoic acid (nonanoic acid) is a naturally occurring medium chain organic acid. It is oily, colorless fluid, which at lower temperature becomes solid. It has a faint odor compared to butyric acid and is almost insoluble in water. The primary use of nonanoic acid has been as a non-selective herbicide. Scythe (57% nonanoic acid, 3% related organic acids and 40% inert material) is a broad-spectrum post-emergence or burn-down herbicide produced by Mycogen/Dow Chemicals. The herbicidal mode of action of nonanoic acid is due first to membrane leakage during darkness and daylight and second to peroxidation driven by radicals originating during daylight by sensitized chlorophyll displaced from the thylakoid membrane (Lederer, et. al., 2004).
Chadeganipour and Haims (2001) showed that the minimum inhibitory concentration (MIC) of medium chain organic acids to prevent growth of M. gypseum was 0.02 mg/ml capric acid and for nonanoic acid 0.04 mg/ml on solid media and 0.075 mg/ml capric acid and 0.05 mg/ml nonanoic in liquid media. These acids were tested independently and not as a mixture. Hirazawa, et. al. (2001) reported that nonanoic acid as well as C6 to C10 organic acids were effective in controlling the growth of the parasite, C. irritans, and that C8, C9 and C19 organic acids were more potent. It was found that Trichoderma harzianum, a biocontrol for cacao plants, produces nonanoic acid as one of many chemicals, which was effective in controlling the germination and growth of cacao pathogens (Aneja, et. al., 2005).
Several US patents disclose the use of nonanoic acids as fungicides and bactericides: US Patent Application #2004/026685) discloses a fungicide for agricultural uses that is composed of one or more fatty acids and one or more organic acids different from the fatty acid. In the mixture of the organic acids and the fatty acids, the organic acid acts as a potent synergist for the fatty acid to function as a fungicide. U.S. Pat. No. 5,366,995 discloses a method to eradicate fungal and bacterial infections in plants and to enhance the activity of fungicides and bactericides in plants through the use of fatty acids and their derivatives. This formulation consists of 80% nonanoic acid or its salts for the control of fungi on plants. The fatty acids used are primarily C9 to C18. U.S. Pat. No. 5,342,630 discloses a novel pesticide for plant use containing an inorganic salt that enhance the efficacy of C8 to C22 fatty acids. One of the examples shows a powdered product with 2% nonanoic acid, 2% capric acid, 80% talc, 10% sodium carbonate and 5% potassium carbonate. U.S. Pat. No. 5,093,124 discloses a fungicide and arthropodice for plants comprising of alpha mono carboxylic acids and their salts. The fungicide consists of the C9 to C10 fatty acids, partially neutralized by an active alkali metal such as potassium. The mixture described consists of 40% active ingredient dissolved in water and includes 10% nonanoic, 10% capric acid and 20% coconut fatty acids, all of which are neutralized with potassium hydroxide. U.S. Pat. No. 6,596,763 discloses a method to control skin infection comprised of C6 to C18 fatty acids or their derivatives. U.S. Pat. No. 6,103,768 and U.S. Pat. No. 6,136,856 discloses the unique utility of fatty acids and derivatives to eradicate existing fungal and bacterial infections in plants. This method is not preventive but showed effectiveness in already established infections. Sharpshooter, a commercially available product, with 80% nonanoic acid, 2% emulsifier and 18% surfactant, is effective against Penicillium and Botrytis spp. U.S. Pat. No. 6,638,978 discloses an antimicrobial preservative composed of a glycerol fatty acid ester, a binary mixture of fatty acids (C6 to C18) and a second fatty acid (C6 to C18) where the second fatty acid is different from the first fatty acid for preservation of food. WO 01/97799 discloses the use of medium chain fatty acids as antimicrobial agents. It shows that an increase of the pH from 6.5 to 7.5 increased the MIC of the short chain (C6 to C18) fatty acids.
Nonanoic acid is used as a component of a food contact surface sanitizing solution in food handling establishments. A product from EcoLab consists of 6.49% nonanoic acid as active ingredient to be use as a sanitizer for all food contact surfaces (12CFR178.1010 b). The FDA has cleared nonanoic acid as a synthetic food flavoring agent (21CFR172.515) as an adjuvant, production aid and sanitizer to be used in contact food (12CFR178.1010 b), and in washing or to assist in lye peeling of fruits and vegetables (12CFR173.315). Nonanoic acid is listed by the USDA under the USDA list of Authorized Substances, 1990, section 5.14, Fruit and Vegetable Washing Compounds.